Cryptococcus neoformans (C. neoformans) is an important cause of life-threatening opportunistic central nervous system (CNS) infection, mainly in HIV-1-infected individuals, but the pathogenesis of C. neoformans CNS infection remains incompletely understood. C. neoformans CNS infection remains a leading contributor to the early mortality following antiretroviral therapy in HIV-1-infected individuals. The available repertoire of antifungal drugs against C. neoformans is limited in number, and the newly developed echinocandins that target fungal ?-1,3-glucan synthase are ineffective against Cryptococcus species. These findings indicate that a new approach is needed for investigating the pathogenesis, prevention and therapy of C. neoformans CNS infection. Several lines of evidence from human cases and experimental animal models of cryptococcal CNS infection indicate that C. neoformans penetration into the brain follows fungemia, and cerebral capillaries are the portal of entry into the brain. Since the entry of C. neoformans into the brain occurred in the cerebral microvasculature, we developed the in vitro blood-brain barrier model with human brain microvascular endothelial cells (HBMEC) to study C. neoformans penetration of the blood-brain barrier. Our HBMEC monolayer, upon cultivation on collagen-coated Transwell inserts, exhibits spatial organization of tight and adherens junction proteins as well as a polarized monolayer, a unique property of the blood-brain barrier endothelial cells. We have shown that C. neoformans strains exhibit the ability to traverse the primary HBMEC monolayer and penetrate into the brain in the animal models of experimental C. neoformans CNS infection. We, therefore, used C. neoformans traversal of the primary HBMEC monolayer as a new approach for discovery of targets affecting C. neoformans penetration into the brain. This R21 application is to capitalize on our discovery of the novel targets affecting C. neoformans traversal of the HBMEC monolayer for investigating their contribution to the pathogenesis, prevention and therapy of C. neoformans CNS infection. Characterization of such targets will demonstrate a novel strategy exploited by C. neoformans for penetration of the blood-brain barrier, and the information will help in development of a new approach for prevention and therapy of C. neoformans CNS infection.